Xenotransplantation offers the best near-term hope for satisfying the critical limitation imposed on the field of transplantation today by the severe shortage of cadaveric allogeneic organs. Our studies in the previous Project period of this Program Project grant have made considerable progress, and organ survivals of both heart and kidney xenotransplants from pigs to baboons are now measured in months rather than days. A major factor in this improved survival has been the development of a new, knock-out strain of miniature swine (GalT-KO), which do not express the Gal epitope on their cells, thus avoiding the severe rejection previously caused by natural anti-Gal antibodies. We have also shown that the greatest improvement in organ survivals using these new donors is observed using protocols designed to induce tolerance. Nevertheless, additional improvements will be required before this technology is ready for clinical application. This renewal application brings together five Projects devoted to the most promising approaches and the most pressing problems in this field of research: 1) tolerance induction through vascularized thymic transplantation;2) tolerance induction through mixed chimerism;3) modeling of tolerance in mice with human immune systems;4) Viral pathogenesis in Xenotransplantation;and 5) thromboregulatory barriers to Xenotransplantation. All of these Projects-are highly interactive, and utilize numerous shared resources, many of which will be made available through the large animal core. The work will be carried out in a unique environment, which provides interactions with scientists working in basic and cellular immunology and working on relevant small and large animal models in the same research center, as well as with clinicians committed to taking new therapies to clinical applications. PROJECT 1: Use of GalT-KO Vascularized Thymic Transplantation for the Induction of Xenogeneic Tolerance in Baboons (Yamada, K.) PROJECT 1 DESCRIPTION (provided by applicant): The overall objective of Project 1 is to induce durable tolerance across a pig-to-baboon xenogeneic barrier through transplantation of vascularized thymic grafts. For this purpose, we will: 1) optimize our immunosuppressive drug regimen: We have demonstrated previously that vascularized thymic grafts support thymopoiesis and induce transplant tolerance across fully mismatched allogeneic barriers. In this xenogeneic model, we will attempt to optimize the immunomodulatory treatment regimen required to achieve durable thymus engraftment;2) determine whether the engrafted thymus can induce tolerance to a kidney xenograft from an inbred donor animal genetically identical to the thymus donor. If successful, the strategy will be extended to simultaneous thymus plus kidney xenotransplantation;and 3) elucidate the mechanism of donor thymus-induced tolerance, using in vitro assays and focusing on the specificity of tolerance across this species barrier. These studies will be highly interactive with other Projects in this PPG. Thus, the optimal treatment regimen will be modified, if necessary, on the basis of results from a mouse model of pig-to-primate tolerance through thymus transplantation being investigated by Sykes and colleagues in Project 3. If eventual antibody-mediated rejection cannot be effectively prevented through thymic transplantation, we will attempt to induce B-cell tolerance by combining thymic transplantation with hematopoietic transplantation, being studied in Project 2. We will also collaborate with Project 5 on the mechanism of any post-transplant coagulation disorders observed. Together, we hope these studies will provide new insights into the potential role of thymic transplantation in the induction of transplantation tolerance.